Deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) are macromolecules built up from simple monomeric subunits called nucleotides. The nucleotide has the following three components: 1) a cyclic five-carbon (pentose) sugar (deoxyribose for DNA, and ribose for RNA), 2) a nitrogenous base of either purine or pyrimidine derivation, covalently attached to the I′-carbon atom of the sugar by a N-glycosylic bond, and 3) a phosphate attached to the 5′ carbon of the sugar by a phosphodiester bond. The purines are adenine (A) and guanine (G), while the pyrimidines are cytosine (C) and thymine (T) for DNA, and cytosine (c) and uracil (U) for RNA.
The nucleotides of DNA are called deoxyribonucleotides, whereas those of RNA are called ribonucleotides. Each nucleotide contains both a specific and a nonspecific region. The phosphate and sugar groups are the nonspecific portions of the nucleotide, while the purine and pyrimidine bases make up the specific portion. Nucleotides are joined to one another linearly by a chemical bond between atoms in the nonspecific regions to form polynucleotides. The linkage, called a phosphodiester bond, is between a phosphate group and a hydroxyl group on the sugar component.
The most important feature of DNA is that it usually consists of two complementary strands coiled about one another to form a double helix. There are ten nucleotides on each chain for every turn of the helix. The two chains are joined together through a combination of van der Waals forces and hydrogen bonds between the purine and pyrimidine base pairs on complementary strands. This base pairing is so specific that adenine binds only to thymine and guanine only to cytosine. This base pairing provides stabilization by hydrogen bonding between complementary bases.
Furthermore, this specificity of base pairing is what permits the transmission of genetic information from one generation to another. When cell replication occurs, the DNA double helix unwinds, and two new complementary DNA strands are formed. The sequence of bases (A, G, T, and C) in a strand of DNA specifies which amino acids are assembled in what order to form proteins. Each amino acid is encoded by a three base sequence of nucleotides, known as a codon; the correlation between each amino acid and its codon is known as the genetic code. The code is degenerate in that several different codons encode the same amino acid.
This base-pairing interaction can be mimicked in vitro by first denaturing the DNA and then allowing it to re-form. DNA is denatured in an aqueous solution by heating to about 100° C. (or pH>13). This disrupts the bonds between the two complementary strands dissociating the double helix into two single strands. These single strands will re-form into a DNA double helix if kept for a prolonged period of time at 65° C. by a process called DNA renaturation or hybridization. Similar hybridization reactions will occur between any two single stranded nucleic acid chains (DNA:DNA, RNA:RNA, or DNA:RNA), provided that they have a complementary nucleotide sequence.
The enormous specificity of this hybridization reaction allows any single-stranded sequence of nucleotides to be labeled with a radioisotope or fluorophore and used as a probe to find a complementary partner strand. Probes of this type are widely used to detect the nucleic acids corresponding to specific genes in situ, by a procedure called in situ hybridization. In situ hybridization is only an example of many other biochemical applications which use base pair specificity.
A complementary artificial DNA/RNA analog has been synthesized and named peptide nucleic acid (PNA)(Nielsen, et al., Science 254:1496-1500, 1991). A representative PNA is a 2-aminoethyl glycine linked by a methylenecarbonyl linkage to one of the four bases (A, G, T, or C) found in DNA. Like amino acids, these molecules have amino and carboxyl termini. Unlike nucleotides, these molecules lack pentose sugar phosphate groups. These properties allow peptide nucleic acids to hybridize to complementary RNA or DNA with higher affinity and specificity than corresponding nucleotides.
In the art, there are several known nucleic acid analogs having nucleobases bound to backbones other than the naturally-occurring ribonucleic acids or deoxyribonucleic acids. These nucleic acid analogs have the ability to bind to nucleic acids with complementary nucleobase sequences. Among these, the peptide nucleic acids (PNAs), as described, for example, in WO 92/20702, have been shown to be useful as therapeutic and diagnostic reagents. This may be due to their generally higher affinity for complementary nucleobase sequence than the corresponding wild-type nucleic acids.
PNAs are compounds that are analogous to oligonucleotides, but differ in composition. In PNAs, the deoxyribose backbone of oligonucleotide is replaced by a peptide backbone. Each subunit of the peptide backbone is attached to a naturally-occurring or non-naturally-occurring nucleobase. One such peptide backbone is constructed of repeating units of N-(2-aminoethyl)glycine linked through amide bonds.
PNAs bind to both DNA and RNA and form PNA/DNA or PNA/RNA duplexes. The resulting PNA/DNA or PNA/RNA duplexes are bound tighter than corresponding DNA/DNA or DNA/RNA duplexes as evidenced by their higher melting temperatures (Tm). This high thermal stability of PNA/DNA(RNA) duplexes has been attributed to the neutrality of the PNA backbone, which results elimination of charge repulsion that is present in DNA/DNA or RNA/RNA duplexes. Another advantage of PNA/DNA(RNA) duplexes is that Tm is practically independent of salt concentration. DNA/DNA duplexes, on the other hand, are highly dependent on the ionic strength.
Homopyrimidine PNAs have been shown to bind complementary DNA or RNA forming (PNA)2/DNA(RNA) triplexes of high thermal stability (Egholm et al., Science, 1991, 254, 1497; Egholm et al., J. Am. Chem. Soc., 1992, 114, 1895; Egholm et al., J. Am. Chem. Soc., 1992, 114, 9677).
In addition to increased affinity, PNAs have increased specificity for DNA binding. Thus, a PNA/DNA duplex mismatch show 8° to 20° C. drop in the Tm relative to the DNA/DNA duplex. This decrease in Tm is not observed with the corresponding DNA/DNA duplex mismatch (Egholm et al., Nature 1993, 365, 566).
A further advantage of PNAs, compared to oligonucleotides, is that the polyamide backbone of PNAs is resistant to degradation by enzymes.
The major advantages of PNAs over DNA (or RNA) for use as in situ hybridization probes are their: (1) higher thermal stability, (2) higher specificity, and (3) resistance to degrading enzymes. The neutral backbone of the PNAs gives to PNA:DNA duplexes (or PNA:RNA) a higher thermal stability compared to DNA:DNA (or RNA:RNA) duplexes. This stronger binding is attributed to the lack of charge repulsion between the PNA strand and the DNA (or RNA) strand. Also, PNAs show a greater specificity in binding to complementary DNA (or RNA). As a result a PNA/DNA mismatch is more destabilizing than a mismatch in a DNA/DNA duplex. PNA oligomers are also resistant to degradation by proteases and nucleases since their polyamide backbone with nucleobase side chains is not a combination easily recognized by these enzymes. This extends the lifetime of any system or device in which PNAs are used.
The object of the present invention is to provide a method for the immobilization of labeled PNAs onto solid surfaces for use in hybridization, purification, biosensing, and other biochemical applications.